Despite effective
antimicrobial chemotherapy, bacterial meningitis is still associated with
a high rate of mortality and neurological sequelae. Without antibacterial
treatment the mortality rate is about 90 %, by use of bactericidal antibacterials
it still is 20 to 25 % (1, 2). Surviving patients are often affected by
neurological sequelae such as hearing loss, epileptic seizures, and paresis
(2). Following the onset of antibacterial treatment a burst of meningeal
inflammation is frequently observed (3, 4). This inflammatory reaction
occurs despite rapid sterilisation of the cerebrospinal fluid (CSF). It
is considered to be caused by a rapid release of bacterial components
induced by the bacteriolytic action of the antibacterials (5). ß-lactam
antibiotics are rapidly bactericidal. As the main aim of chemotherapy
in bacterial meningitis is the sterilisation of the CSF (6), ß-lactam
antibiotics are used for standard therapy. Their mechanism of antibacterial
action is affecting bacterial cell wall synthesis. They are acting by
lysing the bacterial cells. Thus, the therapy leads to a strong release
of bacterial subcomponents as a result of cell lysis. Many of the subcomponents
such as lipopolysaccharides in case of Gram-negative bacteria (7) or peptidoglycan
fragments and teichoic and lipoteichoic acids in case of Gram-positive
bacteria (8) are pro-inflammatory in animal experiments. The high amount
of released bacterial subcomponents can induce the secondary inflammatory
reaction and result in a deleterious outcome for the patient (8, 3).

In the brain,
the released bacterial subcomponents induce the production of pro-inflammatory
cytokines such as tumor necrosis factor alpha (TNF-alpha) and interleukin
1b (IL-1ß). These cytokines are triggering the expression of selectin
molecules on the endothelial cell surfaces which enables neutrophils to
bind to the endothelial cells. Continuous exposure to TNF-alpha and IL-1ß
induces the endothelial cells to release IL-8. IL-8 causes a shift from
the expression of selectin molecules to the expression of integrins. This
enables the neutrophils to traverse the capillary endothelium. In the
CSF, inflammatory cytokines stimulate the neutrophils to produce molecules
such as radicals that contribute to the impairment of the blood brain
barrier (5). The inflammatory reaction can result in brain oedema and
increased intracranial pressure (5). In humans, 20 to 30 % of the meningitis
patients that do not survive die from brain herniation (2). Furthermore,
heat-inactivated rough pneumococci and pneumococcal cell walls produced
direct toxicity on microglial cells and astrocytes cultured in vitro
and neurons co-cultured with glial cells (9, 10). Several approaches were
made to improve the therapy of bacterial meningitis. One approach is to
mitigate the deleterious inflammatory reaction by administering immunosupressive
drugs such as glucocorticoids prior to antibacterial treatment. In an
animal model of E. coli-meningitis this therapy was shown to reduce
several parameters of inflammation and the development of brain oedema
in rabbits (6, 7, 3). Children suffering from bacterial meningitis - mainly
H.-influenzae-meningitis - were treated with dexamethasone
adjunctively and showed significantly reduced frequency of hearing loss
and overall neurological sequelae compared to a group of patients only
treated with the antibacterial (11). In adults there is a lack of randomised
studies documenting a beneficial effect from dexamethasone. Whether glucocorticoids
are beneficial in adults and in children suffering from meningitis caused
by other pathogens is still a matter of debate. Furthermore, animal experiments
suggest that dexamethasone may increase neuronal damage in the dentate
gyrus of the hippocampal formation (12). Another effect of an immunomodulating
therapy is that the blood-brain-barrier tends to be less disturbed than
without this treatment. However, in meningeal inflammation a disturbed
blood brain barrier facilitates the entry of hydrophilic ß-lactam-antibiotics
into the central nervous compartments and assure a high concentration
in the CSF required for effective and rapid killing of the bacteria. Treatment
with dexamethasone decreases the concentration of hydrophilic antibacterials
in the CSF in meningitis and affects CSF sterilisation in a negative
way, particularly in the case of pathogens with a decreased sensitivity
to antibacterials (13, 14).

A therapeutic
approach that circumvents immunosuppression is to decrease the liberation
of bacterial pro-inflammatory cell wall components into the CSF. Therefore
antibacterials must be selected which kill the bacteria effectively but
do not lyse them or at least delay lysis. Then the killed bacteria
remain normally shaped and can be phagocytosed without releasing great
quantities of proinflammatory subcomponents.
Promising antibacterials that may release smaller quantities of proinflammatory
components than ß-lactam antibiotics are those with an antibacterial
mechanism not acting on the cell wall synthesis.In vitro, the release of endotoxin during antibacterial therapy
by Gram-negative bacteria is drug- and dose-dependent (15, 16, 17, 18).
The overall amount of endotoxin released from E.coli cultures treated
with ceftazidime and ciprofloxacin was similar but in serial investigation
the ciprofloxacin-treated cells released only 12,7 % of the lipopolysaccharide
within the first hour of exposure, whereas the ceftazidime-treated bacteria
released 61,9 % in this time period (17). The TNF-alpha and IL-6 levels
produced by stimulation of whole blood with antibacterially treated bacterial
cultures were significantly higher with ceftazidime- and ciprofloxacin-treated
cultures than after imipenem and gentamicin treatment. The levels corresponded
to the amount of released endotoxin (16).

Treatment of
Streptococcus-pneumoniae-cultures with ceftriaxone (ß-lactam)
and rifampin (ansamycin) lead to effective killing of the bacteria. However,
the scanning electron microscopy revealed obvious differences between
both cultures (Fig. 1). The ceftriaxone-treated cells showed blebs and
filaments whereas the rifampin-treated were normally shaped.
Recently, we were able to quantify the release of proinflammatory components
from Streptococcus pneumoniae (19). The most potent proinflammatory
molecules of this species are the teichoic and lipoteichoic acids and
the peptidoglycan fragments (8). The peptidoglycan fragments are not homogeneous
in structure. On the other hand, in pneumococci there is the unique situation
that the polysaccharide part of both, teichoic and lipoteichoic acid,
is identical.
As these molecules are proinflammatory and ubiquitous in all pneumococcal
serotypes they are ideal antigens for an enzyme linked immunosorbent assay
(ELISA). Using this kind of assay we were able to detect pneumococcal
teichoic and lipoteichoic acid in bacterial culture supernatants
down to 0.8 ng/mL.

With this ELISA we investigated
the release of teichoic and lipoteichoic acids by drugs belonging to different
classes of antibacterials. A pathogenic strain of S. pneumoniae
(type 3) was grown in vitro. After resuspension in fresh medium, drugs
were added during the logarithmic phase of growth. In the first
set of experiments, ceftriaxone, rifampin, rifabutin, quinupristin-dalfopristin,
trovafloxacin, or meropenem at concentrations of 10 µg/mL each were added
to the bacterial suspension. The uniformly high concentration of
10 µg/mL was applied to assure a maximum bactericidal effect for all drugs
studied. Samples for the detection of free lipoteichoic acid and teichoic
acid were collected every hour up to 12 h after the addition of drugs.
In these experiments (Fig. 2), the bactericidal rates of ceftriaxone,
meropenem, quinupristin-dalfopristin, and trovafloxacin were slightly
higher than those of rifabutin and rifampin. However, the differences
did not reach statistical significance.
The amount of teichoic and lipoteichoic acids detected in the supernatants
of the pneumococcal cultures revealed significant differences depending
on the antibacterial. Compared to ceftriaxone which is used for standard
therapy, only meropenem treatment did not significantly reduce the concentration
of teichoic and lipoteichoic acid in the supernatant. Meropenem is a carbapenem
belonging to the class of ß-lactam antibiotics and it also acts
on cell wall synthesis. The other four antibacterials tested exhibit other
modes of action. They are inhibitors of the DNA gyrase (trovafloxacin),
of the bacterial RNA-polymerase (rifabutin, rifampin), and of protein
synthesis (quinupristin-dalfopristin), respectively. In the supernatants
of the bacterial cultures treated with these four antibacterials, significantly
lower concentrations of teichoic and lipoteichoic acids were found during
the 12 h lasting experiment. The only exception represented the 10 and
12 h sample from trovafloxacin-treated cultures.

The
in vitro experiments showed that despite similar bactericidal rate
the release of teichoic and lipoteichoic acid to the culture supernatant
was significantly lower in cultures treated with antibacterials not primarily
acting on cell wall synthesis than in cultures treated with the standard
therapy antibiotic, the ß-lactam ceftriaxone.

Lowering
the antibacterial concentration in the in vitro experiments led
to increasing release of teichoic and lipoteichoic acids (Tab. 1). This
effect was observed with all six antibacterials tested. Only rifabutin
and rifampin caused, even at the MIC, relatively low release of bacterial
subcomponents compared to the untreated control. These in vitro data do
not support the concept of using a low first antibiotic dose to prevent
the release of proinflammatory cell wall components (19).

In
the rabbit model of pneumococcal meningitis several antibacterials tested
in the in vitro experiments were used in vivo. Teichoic and
lipoteichoic acids as well as several parameters of inflammation and neurological
sequelae were determined for each treatment group compared to the standard
therapy with ceftriaxone.

Table
1.
Release of LTA and TA by S. pneumoniae type 3 during treatment
with antibacterial agents at different concentrations

In
a first set of experiments the quinolone trovafloxacin was investigated
as an antibacterial for pneumococcal meningitis (20). Animals were injected
intracisternally with pneumococci that were precedingly treated with antibacterials
in vitro. Following injection of ceftriaxone-treated bacterial cultures
consistently higher CSF leukocyte counts (median 2568/µL versus 543/µL at
6 h; p = 0.03; 4560/µL versus 2207/µL at 18 h, p = 0.03) were observed than
with trovafloxacin-treated bacterial cultures.
After intracisternal injection of live pneumococci and subsequent antibacterial
treatment, the amount of teichoic and lipoteichoic acids in the CSF was
lower in the trovafloxacin-treated group than in the ceftriaxone-treated
group. At two hours after initiation of antibacterial therapy the difference
was significant (21). The IL-1ß concentration in the CSF was lower
at two and five hours after initiation of antibacterial therapy in the trovafloxacin-treated
group of animals than in the ceftriaxone-treated group (455 pg/mL and 2921
pg/mL versus 1399 pg/mL and 4302 pg/mL; P = 0.02 at 2 hours, difference
not significant at 5 h). The TNF concentration in the CSF was also significantly
lower than in the standard group two hours after initiation of the antibacterial
treatment (26 U/mL versus 141 U/mL; P = 0.02)). However, the maximum
TNF and IL-1ß concentrations were only delayed, but not decreased in comparison
to ceftriaxone. The lactate increase in CSF during therapy was significantly
lower in the trovafloxacin-treated group. Yet, the parameters of neuronal
destruction (CSF neuron specific enolase [NSE]
and density of neuronal apoptoses in the dentate gyrus; 12) were nearly
identical. This indicated that trovafloxacin delayed but did not inhibit
the inflammatory burst induced by the initiation of antibiotic therapy and
did not influence parameters of neuronal damage.

Schmidt
et al. (22) tested the new quinolone moxifloxacin for antibacterial treatment
of pneumococcal meningitis in the experimental animal model. At a concentration
of 10 mg/kg/h it was found to be as bactericidal as the ceftriaxone standard
(10 mg/kg/h) whereas it was less effective at 2.5 mg/kg/h. Following antibacterial
treatment the TNF release was delayed versus treatment with ceftriaxone
reaching its maximum at 17 h versus 14 h. The CSF leukocyte density, lactate
and protein were similar in both groups. No significant difference in CSF
NSE concentration was observed. Despite a reduced release of proinflammatory
bacterial subcomponents in vitro and a delayed production of TNF
in vivo the inflammatory reaction following antibacterial treatment of pneumococcal
meningitis could not be avoided.

In the CSF of animals treated with rifabutin (5 mg/kg/h) in the rabbit model
of pneumococcal meningitis, significantly lower concentrations of teichoic
and lipoteichoic acid than in the CSF of ceftriaxone (10 mg/kg/h)
treated animals were found at two, five and eight hours after initiation
of antibacterial therapy while the bactericidal rates of both antibacterials
were comparable (21). Consistently, the TNF concentration in the CSF of
rifabutin-treated rabbits was significantly lower than in the CSF of ceftriaxone-treated
animals at two and five hours after initiation of the antibacterial therapy
(23). No difference was found after 12 hours (Fig. 3). Other parameters
of inflammation such as CSF lactate, protein, and IL-1ß were found to be
similar to those obtained with ceftriaxone-treated rabbits. The median leukocyte
density was lower 2 h after initiation of therapy, the difference not reaching
statistical significance. The CSF concentration of NSE and the density of
apoptotic neurones in the dentate gyrus did not differ significantly (23).

In
conclusion, rifabutin was rapidly bactericidal in vivo and caused a lower
release of proinflammatory bacterial subcomponents and a lower level of
production of TNF. The probable reason is the dissociation between bacterial
killing and lysis. As trovafloxacin, rifabutin did not diminish parameters
of neuronal damage in comparison to ceftriaxone.

In the CSF of rabbits treated with a bolus dose or a continuous infusion
of the streptogramin quinupristin-dalfopristin, respectively, the teichoic
and lipoteichoic acid concentrations were lower than in the CSF of animals
treated with ceftriaxone (21, 24). The difference was significant at five
and eight hours after initiation of therapy. The TNF concentrations were
lower, too. The differences were significant at two and five hours after
initiation of antibacterial therapy in both groups and at eight hours after
initiation in the group treated with a bolus of quinupristin-dalfopristin.
The CSF lactate and protein concentrations were similar in all groups. After
12 h of treatment with quinupristin-dalfopristin, however, the NSE concentrations
in the CSF as a parameter of neuronal damage were significantly lower with
both applications than in the ceftriaxone-treated group (Fig. 4). The density
of apoptotic neurones was also lower in quinupristin-dalfopristin-treated
rabbits, yet, the difference did not reach statistical significance (24).

The
bactericidal rate of quinupristin-dalfopristin was lower than that of ceftriaxone,
probably due to low CSF concentrations, in particular, of dalfopristin.
In vitro quinupristin-dalfopristin was as active as ceftriaxone. The very
low release of proinflammatory bacterial components, the low TNF concentrations
produced, and the delayed increase of leukocyte density as well as the significantly
lower CSF NSE values observed, altogether suggest an attenuated inflammatory
response and a reduction of the extent of neurological sequelae. Therefore,
streptogramins should be taken into account as an alternative antibacterial
option, provided that modified dosing regimens or agents reaching higher
CSF concentration and a rapid bactericidal activity in the CSF will be available
in the future.

Antibiotics not primarily affecting cell wall synthesis reduce the concentrations
of proinflammatory pneumococcal sub-components in vitro and in vivo
in comparison with ß-lactam antibiotics following initiation of therapy.
In vivo, the levels of several

parameters
of inflammation and with quinupristin-dalfopristin even the CSF NSE concentration
as a parameter of neurological sequelae were reduced following treatment
with less bacteriolytic antibacterial.

In the mouse model of pneumococcal meningitis we recently demonstrated that
treatment with the ansamycin rifampin reduces the early mortality rate significantly
compared to treatment with ceftriaxone (26 % versus 49 %, P = 0.04; 25).
Correspondingly, the teichoic and lipoteichoic acid levels were lower in
serum samples and pooled CSF of the animals treated with rifampin than in
the specimens of the animals treated with ceftriaxone 8 h after the initiation
of therapy.

In conclusion, the present experiments demonstrate that the use of antibacterials
that act bactericidally without lysing bacteria is a promising approach
toward reducing the secondary inflammatory reaction following antibacterial
treatment and early mortality in bacterial meningitis.